Immunofluorescence staining was performed as described before [16 (link)] with some modification, to detect the expression levels and differences of fibronectin (FN), collagen I (Col I), and α-smooth muscle actin (α-SMA) in the kidneys of five groups (Mod group, Mang-H group, BpV group, and BpV+Mang-H group). The sections of the paraffin-embedded sample were made and blocked with serum-free protein (Dako, Victoria, Australia) and permeabilized for 30 min. Sections were incubated with primary antibodies FN, Col I, α-SMA (Wanleibio, Shijiazhuang, China; 1 : 100 dilution) overnight at 4°C. Sections were washed with TBS 3× 10 min and incubated with species-specific secondary antibodies: Goat Anti-Rabbit IgG H&L (Cy3®) (1 : 1000 dilution, ab6939, Abcam, USA) at room temperature for 1 h. After washing with TBS, the sections were stained with Glycerol Mounting Medium (Abcam) that contained 4,6-diamidino-2-phenylindole (DAPI) and 1,4-diazobicyclo-2,2,2-octane (DABCO). Labelled tissues were visualized using a Leica DM LB2 microscope. Fluorescence images (400x magnification) were captured using NIS-Elements 4.13 (Nikon, Japan) software. The number of fluorescence-positive cells was counted from five representative high-power fields (400x magnification) per tissue section.
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